In mammals, the ability to regenerate limbs and organs is progressively lost during ontogeny and correlates closely with maturation of immune competence. Research in scar-free wound healing, primarily observed in model systems with dysfunctional neutrophils and macrophages, has led to the hypothesis that the immune system dictates the balance between scarring and regeneration. We seek to test this hypothesis by studying a regenerative animal. Among vertebrates, urodele amphibians, such as the axolotl (Ambystoma mexicanum), display a unique and extensive ability for adult regeneration. Axolotls can replace a wide variety of tissues and complex structures including muscle, cartilage, skin, spinal cord, brain, heart, jaw, and limbs. Axolotl also do not get cancer. Mutagens do not induce transformation in axolotls at worst they induce spurious regeneration or a small benign neoplasia. Enhanced immune clearance of damaged tissue by axolotl leukocytes is another hypothesis we hope to test. To do so, we must better define the axolotl hematopoietic system - particularly the myeloid lineages where very little beyond morphology is known. Defining the genetic pathways required in the hematopoietic system for regeneration via a comparative approach contrasting matched murine and axolotl wound healing/regeneration models is one long-term goal that will be facilitated by these studies. These studies will also facilitate definig the role of hematopoietic cells in axolotl cancer immunity by similar comparative approaches replicating well establishes murine models of oncogenesis in axolotl. This proposal seeks to provide the initial characterization of axolotl hematopoietic ontogeny with a focus on myeloid development. A PubMed search for axolotl HSC, macrophages, or neutrophils yields fewer than 25 relevant papers published since 1972. Therefore, the design of this proposal is more empirical using tested methodology from mammalian and zebrafish studies to delineate axolotl hematopoiesis/myelopoiesis. Heavy emphasis will be placed on identifying/generating antibody reagents for hematopoietic specific cell surface markers. In addition, we will use previously successful hematopoietic specific transgenic reporter constructs (Vav-1, gata-1, PU.1 (made myself)), c-myb, Lyz (lysozyme), MRP8 (mac specific), Mpx (myeloperoxidase) and NE (neutrophil elastase) regulatory/promoter constructs) to generate matching axolotl transgenic strains to facilitate comparative studies. We have two Specific Aims: Aim 1. Defining the Axolotl HSC: Ontogeny, Function and Niche. Aim2. Characterization of Macrophages and Neutrophils in Axolotl.